Plant potentiators

ABSTRACT

An object of the present invention is to determine nov 1 means of activating plants: more particularly, means of controlling plant growth, such as means of promoting growth, means of controlling dormancy, means of imparting tolerance against stress for plants (dryness, high or low temperatures, osmotic pressure, etc.), and means of preventing aging. The present inventors have found that the above object can be achieved by providing plant activators containing, as an active ingredient, C 4 -C 24 , ketol fatty acids, in particular, 9-hydroxy-10-oxo-12 (Z),15(Z)-octadecadienoic acid.

TECHNICAL FIELD

The present invention relates to a plant activator.

BACKGROUND ART

Development of techniques for activating plants is a very importantissue for improving supply efficiency of grain plants and garden plants.

Factors for determining growth rate of plants include temperature,light, and nutrients. Conventionally, in order to promote growth ofplants, temperature conditions and sunlight irradiation conditions havebeen controlled in accordance with characteristics of plants to begrown. Along with techniques utilizing temperature or light, manuring isa typical technique for promoting growth of plants. Manuring has exertedreliable effects.

However, the effect of manuring is limited, and even when the amount offertilizers employed is increased, the effect of promoting growth ofplants to a higher level cannot be expected. Employment of excessiveamounts of fertilizers may not only inhibit growth of plants, but alsoinduce contamination of soil.

Particularly, at an early stage of growth of a plant, growth disorderattributed to manuring tends to occur, and therefore manuring isgenerally not performed at this stage.

An object to be achieved by the present invention is to determine meansfor activating plants different from conventional activating means;specifically, means for controlling growth of plants, such as means forpromoting growth of plants, means for preventing dormancy of plants,means for imparting to plants tolerance against stresses (e.g., dryness,high or low temperatures, and osmotic pressure), and means forpreventing aging of plants, to thereby potentiate plants, with theamounts of fertilizers employed being reduced and contamination of soilbeing prevented.

DISCLOSURE OF THE INVENTION

In order to achieve the object, the present inventors have performedextensive studies. As a result, the present inventors have found that,surprisingly, a specific ketol fatty acid exerting “the effect ofpromoting flower bud formation” (see Japanese Patent ApplicationLaid-Open (kokai) No. 11-29410) also exerts “the effect of activatingplants,” which is, in a sense, contrastive to the effect of promotingflower bud formation. The present invention has been accomplished on thebasis of this finding.

Accordingly, the present invention provides a plant activator comprisinga C₄-C₂₄, ketol fatty acid as an active ingredient (hereinafter theactivator may be referred to as “the present plant activator”).

As used herein, the expression “activation of plants” refers tocontrolling plant growth in a certain manner so as to activate ormaintain growth of plants, and encompasses actions for controlling plantgrowth, such as promotion of growth (including increase of size of stemsand leaves and promotion of growth of tubers and tuberous roots),control o dormancy, impartment of tolerance against stresses, andprevention of aging. “Activation of plants” is, in a sense, contrastiveto “promotion of flower bud formation”, described in Japanese PatentApplication Laid-Open (kokai) No. 11-29410. Formation of flower buds isa phenomenon associated with suppressing life activity of plants. Ingeneral, flower buds are formed when growth of plants is inhibited. Asis well known in the horticultural field, when blooming of flowers isdesired, the following operations—which are considered to arrest growthof plants—are performed: (1) the amount of a nitrogenous fertilizeremployed is reduced, (2) the frequency of water sprinkling is reduced,(3) roots are cut, and (4) damage is inflicted to stems. Formation offlowers is a generative Phenomenon at a mature stage of plants fortransmitting their genes to the next generation, and requires a largeamount of energy.

As described above, the aforementioned ketol fatty acid exerting theeffect of promoting flower bud formation quite unexpectedly exerts theeffect of activating plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of evaluation of growth promotion effect ofspecific ketol fatty acid (I) on morning glory.

FIG. 2 shows the results of evaluation of growth promotion effect ofspecific ketol fatty acid (I) on lettuce.

FIG. 3 shows the results of evaluation of growth promotion effect ofspecific ketol fatty acid (I) on broad bean.

FIG. 4 shows the results of evaluation of growth promotion effect ofspecific ketol fatty acid (I) on Eustoma russellianum.

FIG. 5 shows the results of evaluation of growth promotion effect ofspecific ketol fatty acid (I) on cyclamen.

FIG. 6 shows the results of evaluation of growth promotion effect ofspecific ketol fatty acid (I) on digitalis.

FIG. 7 shows the results of evaluation of growth promotion effect ofspecific ketol fatty acid (I) on chrysanthemum.

FIG. 8 shows the results of evaluation of growth promotion effect ofspecific ketol fatty acid (I) on geranium.

FIG. 9 shows the results of evaluation of growth promotion effect ofspecific ketol fatty acid (I) on Primula melacoides.

FIG. 10 shows the results of evaluation of growth promotion effect ofspecific ketol fatty acid (I) on Begonia sempaflorens.

FIG. 11 shows the results of evaluation of growth promotion effect ofspecific ketol fatty acid (I) on Dianthus caryophyllus.

FIG. 12 shows the results of evaluation of growth promotion effect ofspecific ketol fatty acid (I) on Oryza sativa L.

FIG. 13 shows the results of evaluation of growth controlling effect ofspecific ketol fatty acid (I) on Oryza sativa L. in consideration ofpractical culture.

FIG. 14 shows the results of evaluation of dormancy preventive effect ofspecific ketol fatty acid (I) on strawberry.

FIG. 15 shows the results of evaluation of proliferation enhancingeffect of specific ketol fatty acid (I) on hyphae of Pleurotuscitrinopileatus Sing.

FIG. 16 is a photograph showing the results of evaluation of growthpromotion effect of specific ketol fatty acid (I) on carpophore ofLentinu edodes (Berk.) Singer.

BEST MODE FOR CARRYING OUT THE INVENTION

Embodiments of the present invention will next be described.

The present plant activator contains a specific ketol fatty acid as anactive ingredient.

As described above, the ketol fatty acid is a C₄-C₂₄ ketol fatty acid(hereinafter the ketol fatty acid may be referred to as “the specificketol fatty acid”).

Briefly, the specific ketol fatty acid is a C₄-C₂₄ fatty acid having ahydroxyl group of alcohol and a carbonyl group of ketone in themolecule.

In the present invention, preferably, the specific ketol fatty acidcontains the carbon atom constituting the carbonyl group and the carbonatom connected to the hydroxyl group at an α or γ position, in order toexert desired effects of poteniating plants. From this viewpoint,particularly preferably, the carbon atoms are present at an α position.

The specific ketol fatty acid preferably contains one to sixcarbon-carbon double bonds (note: the number of the double bonds doesnot exceed the number of carbon-carbon bonds in the ketol fatty acid),in order to exert desired effects of poteniating plants.

Preferably, the specific ketol fatty acid contains 18 carbon atoms, andtwo carbon-carbon double bonds.

Specific examples of the specific ketol fatty acid include9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid [hereinafter may bereferred to as “specific ketol fatty acid (I)”],13-hydroxy-12-oxo-9(Z),15(Z)-octadecadienoic acid [hereinafter may bereferred to as “specific ketol fatty acid (II)”],13-hydroxy-10-oxo-11(E),15(Z)-octadecadienoic acid [hereinafter may bereferred to as “specific ketol fatty acid (III)], and9-hydroxy-12-oxo-10(E),15(Z)-octadecadienoic acid [hereinafter may bereferred to as “specific ketol fatty acid (IV)].

The chemical formulas of specific ketol fatty acids (I) and (IV) are asfollows.

The chemical formulas of specific ketol fatty acids (II) and (III) aredescribed below in connection with the chemical synthesis method ofthese fatty acids.

Among specific ketol fatty acids, at least some ketol fatty acids areknown as metabolic intermediates of fatty acids in animals and plants,but the role that the ketol fatty acids play directly in plants is notknown.

For example, specific ketol fatty acid (I) is known as an intermediatein a fatty acid metabolic pathway in which α-linolenic acid, which isabundantly present in living organisms, serves as a starting material.However, the specific role that ketol fatty acid (I) plays directly inplants has remained unknown.

The present inventors have found that the aforementioned ketolunsaturated fatty acids related to the present invention exert theeffect of activating plants.

A. Production Method of specific Ketol Fatty Acid

A desired specific ketol fatty acid can be produced by means of a methodin accordance with the specific structure of the fatty acid.

specifically, (1) a specific ketol fatty acid which is known to bepresent in a naturally occurring product can be prepared from theproduct by means of a method in which the naturally occurring product issubjected to extraction and purification (hereinafter the method will bereferred to as an “extraction method”); (2) a specific ketol fatty acidcan be produced by means of a method in which an unsaturated fatty acidis reacted with an enzyme such as lipoxygenase in a manner similar tothat of a fatty acid metabolic pathway in plants (hereinafter the methodwill be referred to as an “enzyme method”); and (3) a desired specificketol fatty acid can be produced by means of a known chemical synthesismethod in accordance with the specific structure of the fatty acid(hereinafter the method will be referred to as a “chemical synthesismethod”).

(1) Extraction Method

Specific ketol fatty acid (I) can be obtained from Lemna paucicostata,which belongs to Lemnaceae, through extraction and purification.

Lemna paucicostata, used as a source material in this extraction methodis a small water plant floating on the surface of a pond or a paddyfield, and each thallus floating on the water forms one root in thewater. Lemna paucicostata is known to have a relatively fast growthrate. The flower thereof is formed on the side of the thallus in whichtwo male flowers containing only one stamen and a female flowercontaining one pistil are enveloped in a small common bract.

The homogenate of Lemna paucicostata is subjected to centrifugation(8,000×g, about 10 minutes), and the fraction obtained by removing thesupernatant from the resultant supernatant and precipitate can be usedas a fraction containing specific ketol fatty acid (I).

As described above, specific ketol fatty acid (I) can be isolated andpurified from the aforementioned homogenate serving as a startingmaterial.

An aqueous solution obtained by floating or immersing Lemna paucicostatain water may be used as a preferred starting material in terms ofpreparation efficiency. No particular limitation is imposed on theaqueous solution, so long as Lemna paucicostata is viable in thesolution.

Specific methods for preparing the aqueous solution are described belowin Examples.

The immersing time is not particularly limited, and may be about two tothree hours at room temperature.

When the starting material of specific ketol fatty acid (I) is preparedby means of the aforementioned method, in consideration of productionefficiency of specific ketol fatty acid (I), Lemna paucicostata ispreferably subjected, in advance, to specific stress which enablesinduction of specific ketol fatty acid (I).

Specific examples of the aforementioned stress include dry stress, heatstress, and osmotic pressure stress.

The dry stress may be imposed on Lemna paucicostata, for example, byallowing Lemna paucicostata to spread on a dry paper filter at lowhumidity (preferably at a relative humidity of 50% or less) and at roomtemperature, preferably at 24-25° C. In this case, the drying time,which varies with the spreading density of Lemna paucicostata to bedried, is about 20 seconds or more, preferably five minutes to fivehours.

The heat stress may be imposed on Lemna paucicostata, for example, byimmersing Lemna paucicostata in hot water. In this case, the temperatureof hot water is determined in accordance with the immersing time. Forexample, when the immersing time is about five minutes, the temperatureof hot water is 40-65° C., preferably 45-60° C., more preferably 50-55°C. Preferably, immediately after the aforementioned heat stresstreatment, Lemna paucicostata is returned to water of ambienttemperature.

The osmotic pressure stress may be imposed on Lemna paucicostata, forexample, by bringing Lemna paucicostata into contact with a solution ofhigh osmotic pressure, such as a sugar solution of high concentration.In the case, when a mannitol solution is used, the sugar concentrationis 0.3 M or more, preferably 0.5-0.7 M. When a 0.5 M mannitol solutionis used, the treatment time is one minute or more, preferably two tofive minutes.

Thus, a desired starting material containing specific ketol fatty acid(I) can be prepared.

No particular limitation is imposed on the strain of Lemna paucicostataserving as a source material of the aforementioned various startingmaterials, but a strain P441 is particularly preferred when specificketol fatty acid (I) is to be produced.

A starting material prepared as described above may be subjected to thebelow-described separation and purification, to thereby produce desiredspecific ketol fatty acid (I).

The separation method employed for producing specific ketol fatty acid(I) from the aforementioned starting material is not limited to thebelow-described example separation methods.

Firstly, the aforementioned starting material is preferably subjected toextraction by use of a solvent, to thereby obtain an extract containingspecific ketol fatty acid (I). Examples of the solvent include, but arenot limited to, chloroform, ethyl acetate, and ethers. Of thesesolvents, chloroform is preferred, since it enables removal ofimpurities in a relatively easy manner.

The oil layer fractions obtained through the solvent extraction arewashed and concentrated by means of a conventionally known method, andthen subjected to high performance liquid chromatography (HPLC) by useof a reversed-phase partition chromatography column such as an ODS(octadecylsilane) column, to thereby identify and isolate a fractionhaving ability to induce flower bud formation, thereby potentiallyisolating specific ketol fatty acid (I) [note: the specific ketol fattyacid is known to have ability to induce flower bud formation (seeJapanese Patent Application Laid-Open(kokai) No. 10-324602)].

In accordance with properties of the starting material, otherconventionally known separation methods, such as ultrafiltration and gelfiltration chromatography, may be employed in combination.

The production process of specific ketol fatty acid (I) by means of theextraction method has been described above. When a desired specificketol fatty acid is present in a plant other than Lemna paucicostata,the fatty acid can be produced by means of a method similar to thatdescribed above or a modification of the aforementioned method.

(2) Enzyme Method

Typical examples of the starting material employed in the extractionmethod include C₄-C₂₄ unsaturated fatty acids having carbon-carbondouble bonds at positions corresponding to those of carbon-carbon doublebonds contained in a desired specific ketol fatty acid.

Examples of the unsaturated fatty acids include, but are not limited to,oleic acid, vaccenic acid, linoleic acid, α-linolenic acid, γ-linolenicacid, arachidonic acid, 9,11-octadecadienoic acid, 10,12-octadecadienoicacid, 9,12,15-octadecadienoic acid, 6,9,12,15-octadecatetraenoic acid,11,14-eicosadienoic acid, 5,8,11-eicosatrienoic acid,11,14,17-eicosatrienoic acid, 5,8,11,14,17-eicosapentaenoic acid,13,16-docosadienoic acid, 13,16,19-docosatrienoic acid,7,10,13,16-docosatetraenoic acid, 7,10,13,16,19-docosapentaenoic acid,and 4,7,10,13,16,19-docosahexaenoic acid.

These unsaturated fatty acids are generally present in animals andplants. The fatty acids may be obtained from animals and plants throughextraction and purification by means of conventionally known methods, ormay be obtained through chemical synthesis by means of conventionallyknown methods. Alternatively, the fatty acids may be commerciallyavailable products.

In the enzyme method, the aforementioned unsaturated fatty acid servingas a substrate is reacted with lipoxygenase (LOX), to thereby introducea hydroperoxy group (—OOH) into the carbon chain of the unsaturatedfatty acid.

Lipoxygenase is an oxidoreductase which introduces molecular oxygen, asa hydroperoxy group, into the carbon chain of an unsaturated fatty acid.As has been confirmed, lipoxygenase is present in animals and plants, aswell as in yeast such as saccharomyces.

For example, the presence of lipoxygenase is recognized in plants suchas angiosperms [specifically, the below-described dicotyledons andmonocotyledons to which the present plant activator can be applied].

Among the aforementioned plants, examples of the particularly preferredorigin of lipoxygenase include soybean, flax, alfalfa, barley, broadbean, lupine, lentil, field pea, potato, wheat, apple, bread yeast,cotton, cucumber, gooseberry, grape, pear, kidney bean, rice,strawberry, sunflower, and tea. Since chlorophyll tends to inhibit theaforementioned activity of lipoxygenase, if possible, lipoxygenase ispreferably obtained from seeds, roots, fruits, etc. of the plants inwhich chlorophyll is not present.

In the present invention, lipoxygenase of any origin may be used, solong as it can introduce a hydroperoxy group into a desired position ofthe carbon chain of an unsaturated fatty acid. However, when specificketol fatty acid (I) is produced, if possible, lipoxygenase whichenables selective oxidation of the carbon-carbon double bond at position9 of linoleic acid or linolenic acid is preferably used.

Typical examples of the selective lipoxygenase include lipoxygenasederived from rice germ [e.g., Yamamoto, A., Fuji, Y., Yasumoto, K.,Mitsuda, H., Agric. Biol. Chem., 44, 443 (1980)].

Preferred examples of the unsaturated fatty acid serving as a substratewith respect to the selective lipoxygenase include linoleic acid andα-linolenic acid.

When an unsaturated fatty acid serving as a substrate is treated withlipoxygenase, enzymatic reaction is preferably allowed to proceed at anoptimum temperature and an optimum pH of the lipoxygenase to beemployed.

Unwanted impurities generated through the aforementioned lipoxygenasereaction process may be easily separated by means of conventionallyknown methods, such as HPLC described above in (1).

Lipoxygenase used herein may be obtained from, for example, theaforementioned plants through extraction and purification by means ofconventionally known methods, or may be a commercially availableproduct.

Thus, a hydroperoxy unsaturated fatty acid can be produced from theaforementioned unsaturated fatty acid.

The hydroperoxy unsaturated fatty acid may be considered an intermediatein the production process of a specific ketol fatty acid by means of theenzyme method.

Examples of the hydroperoxy unsaturated fatty acid include9-hydroperoxy-10(E),12(Z),15(Z)-octadecatrienoic acid, which serves asan intermediate of the aforementioned specific ketol fatty acid (I) andcan be obtained by reacting α-linolenic acid with lipoxygenase.

Of these hydroperoxy fatty acids, the former9-hydroperoxy-10(E),12(Z),15(Z)-octadecatrienoic acid will be called“hydroperoxy fatty acid (a)” in relation to the present invention, andthe latter 13-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid will becalled “hydroperoxy fatty acid (b)” in relation to the presentinvention. The chemical formulas of these hydroperoxy fatty acids aredescribed below.

Subsequently, the hydroperoxy unsaturated fatty acid serving as asubstrate is reacted with allene oxide synthase, to thereby produce adesired specific ketol fatty acid.

Allene oxide synthase is an enzyme having the activity of converting ahydroperoxy group, via epoxidation, into a ketol structure. Similar tothe aforementioned lipoxygenase, allene oxide synthase is present inplants, animals, and yeast. For example, allene oxide synthase ispresent in plants such as angiosperms [specifically, the below-describeddicotyledons and monocotyledons to which the present plant activator canbe applied].

The presence of allene oxide synthase is recognized in plants such asbarley, wheat, corn, cotton, eggplant, flax (e.g., flax seed), lettuce,oat, spinach, and sunflower.

In the present invention, no particular limitation is imposed on theallene oxide synthase employed, so long as it enables formation of anepoxy group through dehydration of the hydroperoxy group at position 9of, for example, the aforementioned9-hydroperoxy-10(E),12(Z),15(Z)-octadecatrienoic acid, to therebyproduce a desired specific ketol fatty acid through nucleophilicreaction of OH⁻.

When the aforementioned allene oxide synthase treatment is performed,enzymatic reaction is preferably allowed to proceed at an optimumtemperature and an optimum pH of the allene oxide synthase to beemployed.

Allene oxide synthase used herein may be obtained from, for example, theaforementioned plants through extraction and purification by means ofconventionally known methods, or may be a commercially availableproduct.

The aforementioned two enzymatic reaction processes may be performedseparately or successively. The aforementioned enzymatic reaction may beallowed to proceed by use of the crude or purified product of theaforementioned enzyme, to thereby produce a desired specific ketol fattyacid. Also, the aforementioned enzyme may be immobilized on a carrier,after which the enzyme substrate may be subjected to column treatment orbatch treatment by use of the thus-immobilized enzyme, to therebyproduce a desired specific ketol fatty acid.

The enzymes employed in the aforementioned two processes may be preparedby means of a genetic engineering technique. Specifically, the enzymescan be produced as follows: genes encoding the enzymes are obtainedfrom, for example, plants through extraction by means of a customarymethod, or the genes are obtained through chemical synthesis on thebasis of the genetic sequence of the enzymes; microorganisms such asEscherichia coli and yeast, animal cultured cells, or plant culturedcells are transformed by use of the above-obtained genes; and therecombinant enzyme protein is expressed in the resultant transformedcells.

When a specific ketol fatty acid is produced through nucleophilicreaction of OH⁻ (described above) after formation of an epoxy group,depending on the reaction of the nucleophile in the vicinity of theepoxy group, a γ-ketol compound is formed in addition to an α-ketolunsaturated fatty acid.

The thus-formed γ-ketol compound can be easily separated from theα-ketol compound by means of a conventionally known separation method,such as HPLC described above in (1).

(3) Chemical Synthesis Method

A specific ketol fatty acid may be produced by means of conventionallyknown chemical synthesis methods.

For example, a saturated carbon chain having, at its one end, a reactivegroup such as an aldehyde group and having, at the other end, a carboxylend group connected to a protective group is synthesized by means of aconventionally known method, and separately, an unsaturated carbon chainhaving, at a desired position, an unsaturated group and having areactive end group is synthesized from a starting material such as anunsaturated alcohol (e.g., cis-3-hexen-1-ol). Subsequently, theresultant saturated hydrocarbon chain and unsaturated carbon chain arereacted with each other, to thereby produce a specific ketol fatty acid.In the aforementioned reactions, the protective group connected to anyend group which does not participate in reaction, and the catalyst forpromoting reaction may be appropriately chosen in accordance with thespecific reaction mode.

More specifically, specific ketol fatty acids may be synthesizedthrough, for example, the below-described processes.

i) Synthesis of Specific Ketol Fatty Acid (I)

Nonanedioic acid monoethyl ester serving as a starting material isreacted with N,N′-carbonyldiimidazole, to thereby yield an acidimidazolide, and subsequently, the acid imidazolide is reduced by use ofLiAlH₄ at low temperature, to thereby synthesize the correspondingaldehyde. The aforementioned starting material may be changed to, forexample, a diol such as 1,9-nonanediol, to thereby synthesize a similaraldehyde.

Separately, cis-3-hexen-1-ol is reacted with triphenylphosphine andcarbon tetrabromide. The resultant bromide is reacted withtriphenylphosphine, and further reacted with chloroacetaldehyde in thepresence of n-BuLi, to thereby form a cis olefin. The cis olefin isreacted with methylthiomethyl p-tolyl sulfone, and then reacted with theabove-synthesized aldehyde in the presence of n-BuLi, to thereby yield asecondary alcohol. The resultant secondary alcohol is protected bytert-butyldiphenylsilyl chloride (TBDPSCl), and subjected to hydrolysisby use of an acid, and then to deprotection, to thereby synthesizedesired specific ketol fatty acid (I).

A brief scheme of an embodiment of the synthesis process of specificketol fatty acid (I) is described below.

ii) Synthesis of Specific Ketol Fatty Acid (II)

Nonanedioic acid monoethyl ester serving as a starting material isreacted with thionyl chloride, and the resultant acid chloride isreduced by use of NaBH₄, to thereby yield an acid alcohol. Subsequently,the free carboxyl group of the resultant acid alcohol is protected, andthe resultant product is reacted with triphenylphosphine and carbontetrabromide. The resultant bromide is reacted with triphenylphosphine,and further reacted with chloroacetaldehyde in the presence of n-BuLi,to thereby form a cis olefin. The cis olefin is reacted withmethylthiomethyl p-tolyl sulfone, and then reacted with, in the presenceof n-BuLi, an aldehyde which has been obtained through PCC oxidation ofcis-3-hexen-1-ol. The resultant product is subjected to deprotection, tothereby accomplish synthesis of desired specific ketol fatty acid (II).

A brief scheme of an embodiment of the synthesis process of specificketol fatty acid (II) is described below.

iii) Synthesis of Specific Ketol Fatty Acid (III)

Methyl vinyl ketone serving as a starting material is reacted withtrimethylsilyl chloride in the presence of LDA and DME. MCPBA andtrimethylaminehydrofluoric acid are added to the resultant silyl etherat a low temperature (−70° C.), to thereby prepare a keto-alcohol.Subsequently, the carbonyl group of the keto-alcohol is protected, andthen, in the presence of triphenylphosphine and trichloroacetone servingas reaction reagents, reaction is allowed to proceed so as to preventaddition of chlorine to the carbon-carbon double bond. The reactionproduct is reacted with formic acid in the presence of tributylarsineand K₂CO₃, to thereby form a trans olefin and then form a chloride.Subsequently, the resultant chloride is reacted with an aldehyde whichhas been obtained through PCC oxidation of cis-3-hexen-1-ol. Theresultant reaction product is bonded to 6-heptenoic acid, and thensubjected to deprotection, to thereby synthesize desired specific ketolfatty acid (III).

A brief scheme of an embodiment of the synthesis process of specificketol fatty acid (III) is described below.

B. The Present Plant Activator

When the present plant activator is applied to a plant, the plant can beactivated. Particularly, the present plant activator exerts plant growthcontrolling effect in various manners so as to activate growth ofplants. “Plant activation effect” and “plant growth controlling effect”will next be described in detail.

(1) Growth Promoting Effect

Application of the present plant activator to a plant can increase thegrowth rate of the plant and improve harvest efficiency of the plant (asdescribed above, increase of size of stems and leaves and promotion ofgrowth of tubers and tuberous roots can be expected). Accordingly, thepresent invention also provides a plant growth promoting agent whichexerts more specific effect; i.e., effect of promoting growth of plants.

When the present plant activator is used for promoting growth of plants,particularly, promotion of growth of plants at an early stage aftergermination—which has been difficult to attain by use of afertilizer—can be attained.

Therefore, when the present plant activator is used as a plant growthpromoting agent, application of the activator is preferably carried outduring seedling or at an early growth stage after germination.

When the present plant activator is merely applied, for example, throughspraying, at an early growth stage after germination, growth of plantsis promoted, and the effect of promoting plant growth is maintained. Asdescribed above, even when the present plant activator is used inexcessive amounts, growth disorder of plants, which occurs when afertilizer is used in excessive amount, is not observed. Therefore,careful consideration of the amount of the activator to be employed isnot necessary.

In the horticultural or agricultural field, instead of distribution ofseeds, which require troublesome handling after delivery, distributionof seedlings is becoming the mainstream. Particularly, in the flowerbusiness, in most cases, gardening amateurs purchase seedlings. When thepresent plant activator is employed before distribution of seedlings,the grown seedlings can be sold.

In the case of rice plants, in general, after seedlings are grown in aseedbed at an early stage, the grown seedlings are planted in a paddyfield. When the present plant activator is applied to seedlings in aseedbed, the growth of the seedlings is promoted, and the number ofstems per strain after planting is increased. In the case of riceplants, the number of spikes per strain can be increased, to therebyenhance harvest efficiency. Similarly, when the present plant activatoris employed, the harvest efficiency of other cereal plants such asbarley and corn or fabaceous plants such as soybean can be enhanced.

The aforementioned properties of the present plant activator aresuitable for increasing the harvest of spinach, lettuce, cabbage,broccoli, or cauliflower.

When the present plant activator is applied to ascomycete orbasidiomycete, the growth of hyphae thereof can be promoted, to therebyincrease the harvest yield of carpophores (mushrooms: for example,Lentinus edodes, oyster mushroom, Lyophyllum decastes, mushroom,Pholiota nameko, Grifola frondosa, and Celtis sinensis). Furthermore,the present plant activator may contribute to establishment of anartificial culture method of mushrooms which at present are difficult toculture artificially (e.g., Tricholoma matsutake).

(2) Dormancy Preventive Effect

When the present plant activator is applied to a plant, the dormancy ofthe plant can be prevented. Specifically, when the present plantactivator is applied to a plant, the “dormancy period” of the plantduring which the growth of the plant is stopped can be reduced orterminated.

Accordingly, the present invention also provides a plant dormancypreventive agent which exerts more specific effect; i.e., effect ofpreventing dormancy of plants.

In the case in which the present plant activator is used as a plantdormancy preventive agent, when the activator is applied to a plantimmediately after germination, the dormancy of the plant can beprevented. Alternatively, the activator may be applied to a dormantplant, to thereby terminate the dormancy of the plant.

(3) Anti-Stress Effect

When the present plant activator is applied to a plant, the plant can beendowed with tolerance against various stresses, such as dry stress,high-temperature stress, low-temperature stress, and osmotic-pressurestress. Briefly, when the present plant activator is employed, there canbe reduced the effect of stresses—which are attributed to climatevariation, induction of germination of seeds, etc.—on cultivated plants,the stresses potentially causing reduction in yield of the plants.

In this sense, the present invention also provides a plant stresssuppressive agent which exerts more specific effect; i.e., effect ofsuppressing stresses imposed on plants.

In the case in which the present plant activator is used as a plantstress suppressor, when the activator is applied to a plant duringgermination of its seed or after germination, the plant can be endowedwith tolerance against stresses.

Application of the present plant activator to a plant may prevent agingof the plant. For example, if the present plant activator is applied toa therophyte plant which is in the period in which the plant becomesweak and is dying, weakening (aging) of the therophyte can be retarded.

No particular limitation is imposed on the upper limit of the amount ofa specific ketol fatty acid—which is an active ingredient of the presentplant activator—applied to plants. Even when a specific ketol fatty acidis applied to plants in a large amount through use of the present plantactivator, negative effects on plants, such as growth inhibition, arebarely observed. In contrast, when a conventionally used plant hormoneagent is excessively applied to plants, considerable negative effects onplants are observed. Therefore, the plant hormone agent must be usedcarefully so as not to be excessively applied to plants. From thisviewpoint, the present plant activator is more advantageous as comparedwith the conventional plant hormone agent.

The lower limit of the amount of the aforementioned specific ketol fattyacid applied to a single plant, which varies with the type and size ofthe plant, is about 1 μM per application.

The amount of a specific ketol fatty acid incorporated into the presentplant activator may be determined in accordance with use of theactivator, the type of a plant to which the activator is to be applied,and the specific product form of the activator. A specific ketol fattyacid may be used as the present plant activator. However, inconsideration of the aforementioned lower limit of the applicationamount of a specific ketol fatty acid, etc., the specific ketol fattyacid is preferably incorporated in an amount of about 0.1-100 ppm, morepreferably about 1-50 ppm, on the basis of the entirety of the plantactivator.

Examples of the product form of the present plant activator includesolutions, solid agents, powders, and emulsions. In accordance with theproduct form, the present plant activator may appropriately containknown pharmaceutically acceptable carrier components and auxiliaryagents for drug production, so long as they do not impede the intendedeffect of the present invention; i.e, plant growth promoting effect.When the present plant activator assumes the form of powders or solidagents, for example, the following carrier components may beincorporated: inorganic substances such as talc, clay, vermiculite,diatomaceous earth, kaolin, calcium carbonate, calcium hydroxide, terraalba, and silica gel; and solid carriers such as flour and starch. Whenthe present plant activator assumes the form of solutions, for example,the following carrier components may be incorporated: liquid carriersincluding water; aromatic hydrocarbons such as xylene; alcohols such asethanol and ethylene glycols; ketones such as acetone; ethers such asdioxane and tetrahydrofuran; dimethylformamide; dimethyl sulfoxide; andacetonitrile. Examples of the auxiliary agents for drug production whichmay be incorporated include anionic surfactants such as alkyl sulfates,alkyl sulfonates, alkyl aryl sulfonates, dialkyl sulfosuccinates;cationic surfactants such as salts of higher aliphatic amines; nonionicsurfactants such as polyoxyethylene glycol alkyl ethers, polyoxyethyleneglycol acyl esters, polyoxyethylene polyalcohol acyl esters, andcellulose derivatives; thickeners such as gelatin, casein, gum arabi;extenders; and binders.

If desired, the present plant activator may further contain typicalplant growth controlling agents, benzoic acid, nicotinic acid,nicotinamide, and pipecolic acid, so long as they do not impede theintended effects of the present invention.

The present plant activator may be applied to various plants in a mannerin accordance with the product form of the activator. For example, thepresent plant activator may be sprayed, dropped, or applied, in the formof solution or emulsion, to the point of growth of a plant, to a portionof the plant, such as stem or leaf, or to the entirety of the plant; ormay be absorbed, in the form of solid agent or powder, in the root ofthe plant via earth. Alternatively, when the present plant activator isused for promoting growth of a water plant such as duckweed, theactivator may be absorbed in the root of the water plant, or theactivator in the form of solid agent may be gradually dissolve in thewater.

The frequency of application of the present plant activator to a plantvaries with the type of the plant or the purpose of application.Basically, desired effects can be obtained through merely a singleapplication. When the activator is applied several times, application ispreferably performed at an interval of one week or more.

No particular limitation is imposed on the type of plants to which thepresent plant activator can be applied, and the activator is effectivefor angiosperms (dicotyledons and monocotyledons), fungi, lichens,bryophytes, ferns, and gymnosperms.

Examples of dicotylendos of angiosperms include Convolvulaceae such asConvolvulus (C. nil), Calystegia (C. japonica, C. hederacea, and C.soldanella), Ipomoea (I. pescaprae, and I. batatas), and Cuscuta (C.japonica, and C. australis), Caryophyllaceae such as Dianthus,Stellaria, Minuartia, Cerastium, sagina, Arenaria, Moehringia,Pseudostellaria, Honkenya, Spergula, Spergularia, Silene, Lychnis,Melandryum, and Cucubalus, and furthermore, Casuarinaceae, Saururaceae,Piperaceae, Chloranthaceae, Salicaceae, Myricaceae, Juglandaceae,Betulaceae, Fagaceae, Ulmaceae, Moraceae, Urticaceae, Podostemaceae,Proteaceae, Olacaceae, Santalaceae, Loranthaceae, Aristolochiaceae,Mitrastemonaceae, Balanophoraceae, Polygonaceae, Chenopodiaceae,Amaranthaceae, Nyctaginaceae, Theligonaceae, Phytolaccaceae, Aizoaceae,Portulacaceae, Magnoliaceae, Trochodendraceae, Cercidiphyllaceae,Nymphaeaceae, Ceracophyllaceae, Ranunculaceae, Lardizabalaceae,Berberidaceae, Menispermaceae, Calycanthaceae, Lauraceae, Papaveraceae,Capparaceae, Brassicaceae (Crusiferae), Droseraceae, Nepenthaceae,Crassulaceae, Saxifragaceae, Pittosporaceae, Hamamelidaceae,Platanaceae, Rosaceae, Fabaceae (Leguminosae), Oxalidaceae, Geraniaceae,Linaceae, Zygophyllaceae, Rutaceae, Simaroubaceae, Meliaceae,Polygolaceae, Euphorbiaceae, Callitrichaceae, Empetraceae, Coriariaceae,Anacardiaceae, Aquifoliaceae, Celastraceae, Staphyleaceae, Icacinaceae,Aceraceae, Hippocastanaceae, Sapindaceae, Sabiaceae, Balsaminaceae,Rhamnaceae, Vitaceae, Elaeocarpaceae, Tiliaceae, Malvales,Sterculiaceae, Actinidiaceae, Theaceae, Clusiaceae (Guttiferae),Elatinaceae, Tamaricaceae, Violaceae, Flacourtiaceae, Stachyuraceae,Possifloraceae, Begoniaceae, Cactaceae, Thymelaeaceae, Elaeagnaceae,Lythraceae, Punicaceae, Rhizophoraceae, Alangiaceae, Melastomataceae,Trapaceae, Onagraceae, Haloragaceae, Hippuridaceae, Araliaceae, Apiaceae(Umbelliferae), Cornaceae, Diapensiaceae, Clethraceae, Pyrolaceae,Ericaceae, Lyrsinaceae, Primulaceae, Plumbaginaceae, Ebenaceae,Symplocaceae, Styracaceae, Oleaceae, Buddlejaceae, Gentianaceae,Apocynaceae, Asclepiadaceae, Polemoniaceae, Boraginaceae, Verbenaceae,Lamiaceae (Labiatae), Solanaceae (Solanum, Lycoperisicon, etc.),Scrophulariaceae, Binoniaceae, Pedaliaceae, Orobanchaceae, Gesneriaceae,Lentibulariaceae, Acanthaceae, Myoporaceae, Phrymaceae, Plantaginaceae,Rubiaceae, Caprifoliaceae, Adoxaceae, Valerianaceae, Dipsacaceae,Cucurbitaceae, Campanulaceae, and Asteraceae (Compositae).

Examples of monocotyledons include Lemnaceae such as Spirodela (S.polyrhiza), and Lemna (L. paucicostata, and L. trisulcaca), Orchidaceaesuch as Cattleya, Cymidium, Dendrobium, Phalaenopsis, Vanda,Paphiopedilum, and Oncidium, Typhaceae, Sparganiaceae, Potamogetonaceae,Najadaceae, Scheuchzeriaceae, Alismataceae, Hydrocharitaceae,Triuridaceae, Poaceae (Gramineae) (Oryza, Hordeum, Triticum, Secale,Zea, etc.), Cyperaceae, Arecaceae (Palmae), Araceae, Eriocaulaceae,Commelinaceae, Pontederiaceae, Juncaceae, Stemonaceae, Liliaceae(Asparagus, etc.), Amaryllidaceae. Dioscoreaceae, Iridaceae, Musaceae,Zingiberaceae, Cannaceae, and Burmanniaceae.

EXAMPLES

The present invention will next be described in detail by way ofExamples, which should not be construed as limiting the inventionthereto.

Production Example Production of Specific Ketol Fatty Acid (I)

Specific ketol fatty acid (I)[9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid] was produced bymeans of an enzyme method as follows.

1. Preparation of Rice-Germ-Derived lipoxygenase

Rice germ (350 g) was washed with petroleum ether, defatted, and thendried. The resultant rice germ (250 g) was suspended in a 0.1 M acetatebuffer solution (pH 4.5) (1.25 L), and the resultant suspension washomogenized.

Subsequently, the resultant homogenized extract was subjected tocentrifugation at 16,000 rpm for 15 minutes, to thereby yield asupernatant (0.8 L). Ammonium sulfate (140.8 g) (30% saturation) wasadded to the supernatant, and the resultant mixture was allowed to standat 4° C. overnight. Thereafter, the mixture was subjected tocentrifugation at 9,500 rpm for 30 minutes, to thereby yield asupernatant (0.85 L). Ammonium sulfate (232 g) (70% saturation) wasadded to the supernatant, after which the resultant mixture was allowedto stand at 4° C. for five hours.

Subsequently, the mixture was subjected to centrifugation at 9,500 rpmfor 30 minutes, to thereby yield a precipitate. The above-yieldedprecipitates (fractions obtained from the rice germ extract throughaddition of ammonium sulfate (30-70% saturation) were dissolved in anacetate buffer solution (pH 4.5) (300 mL), and then heated at 63° C. forfive minutes. Thereafter, the precipitate was removed, and thesupernatant was subjected to desalting through dialysis (3 L×3) by useof an RC dialysis tube (Pore 4, product of Spectrum: MWCO12,000-14,000), to thereby yield a crude solution containing desiredrice-germ-derived lipoxygenase.

2. Preparation Flaxseed-Derived Allene Oxide Synthase

Acetone (250 mL) was added to flaxseeds (200 g) purchased from IchimaruPharcos Co., Ltd. The resultant mixture was homogenized (20 s×3), andthe resultant precipitate was subjected to filtration by use of aperforated plate funnel, to thereby remove the solvent.

Subsequently, the precipitate was again suspended in acetone (250 mL),and the suspension was homogenized (10 s×3), to thereby yield aprecipitate. The precipitate was washed with acetone and ethyl ether,and then dried, to thereby yield flaxseed powder (150 g).

The thus-yielded flaxseed powder (20 g) was suspended in a 50 mMphosphate buffer solution (pH 7.0) (400 mL) under cooling with ice. Theresultant suspension was stirred by use of a stirrer at 4° C. for onehour to extraction.

The resultant extract was subjected to centrifugation at 11,000 rpm for30 minutes. To the supernatant was added ammonium sulfate (105.3 g)(0-45% saturation), and the mixture was allowed to stand for one hourunder cooling with ice. The mixture was further subjected tocentrifugation at 11,000 rpm for 30 minutes, to thereby yield aprecipitate. The precipitate was dissolved in a 50 mM phosphate buffersolution (pH 7.0) (150 mL), and the resultant solution was subjected todesalting through dialysis (3 L×3), to thereby yield a crude solutioncontaining desired flaxseed-derived allene oxide synthase.

3. Preparation of α-Linolenic Acid Sodium Salt

αLinolenic acid serving as a starting material has considerably lowwater solubility. Therefore, in order to cause α-linolenic acid tofunction effectively as an enzyme substrate, an α-linolenic acid sodiumsalt was prepared.

Specifically, sodium carbonate (530 mg) was dissolved in purified water(10 mL), and then heated to 55° C. α-Linolenic acid (product of NacalaiTesque, Inc.) (278 mg) was added dropwise to the resultant solution, andthe mixture was stirred for three hours.

After completion of reaction, the reaction mixture was neutralized withDowex50W-X8 (H⁺ form) (product of Dow Chemical Co.), to thereby yield aprecipitate. The precipitate was subjected to filtration to therebyremove a resin. Subsequently, the precipitate was dissolved in MeOH, andthen the solvent was removed under vacuum.

The thus-obtained product was recrystallized with isopropanol, tothereby yield a desired α-linolenic acid sodium salt (250 mg. 83%).

4. Production of Specific Ketol Fatty Acid (I)

The α-linolenic acid sodium salt yielded above in 3 (15 mg: 50 μmol) wasdissolved in a 0.1 M phosphate buffer solution (pH 7.0) (30 mL). To theresultant solution was added the rice-germ-derived lipoxygenase crudesolution prepared above in 1 (3.18 mL) at 25° C. under oxygen flow, andthe mixture was stirred for 30 minutes. The rice-germ-derivedlipoxygenase crude solution (3.18 mL) was further added to the mixture,and the resultant mixture was stirred for 30 minutes.

After completion of stirring, the allene oxide synthase crude solutionprepared above in 2 (34.5 mL) was added to the lipoxygenase reactionmixture under nitrogen flow, and the resultant mixture was stirred for30 minutes. Thereafter, dilute hydrochloric acid was added to thereaction mixture under cooling by use of ice, to thereby adjust the pHof the mixture to 3.0.

Subsequently, the reaction mixture was subjected to extraction with asolvent mixture of CHCl₃ and MeOH (10:1). The thus-obtained organiclayer was subjected to dehydration through addition of magnesiumsulfate, and the solvent was removed under vacuum and then dried.

The thus-obtained crude product was subjected to HPLC, and a fractioncorresponding to the peak of specific ketol fatty acid (I) (retentiontime: about 16 min.) was obtained. Chloroform was added to thethus-obtained fraction, the separated chloroform layer was washed withwater, and the chloroform was removed by use of an evaporator, tothereby yield a purified product.

In order to confirm the structure of the purified product, the productwas subjected to measurement of ¹H- and ¹³C-NMR spectra by use of aheavy methanol solution.

As a result, in the ¹H-NMR measurement, signals corresponding to an endmethyl group [δ 0.98 (t)], two carbon-carbon double bonds [(δ 5.25,5.40). (δ 5.55, 5.62)], a secondary hydroxyl group [δ 4.09 (dd)], andnumerous methylene groups were observed, and the product was presumed tobe specific ketol fatty acid (I).

Furthermore, the ¹³C-NMR chemical shifts of the product were identicalto the ¹³C-NMR chemical shifts of specific ketol fatty acid (I)described in Japanese Patent Application Laid-Open (kokai) No. 10-324602(in [0054] and (0055], from line 2 of column 13, page 8), the fatty acidbeing produced in “Production Example (extraction method)” described inthe above publication (from last line of column 11, page 7) (see Table1).

Thus, the synthesized product obtained by means of the above enzymemethod was identified as 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoicacid.

TABLE 1

Product synthesized by Standard product means of enzyme method C-1 178.5178.4 C-2 35.7 35.4 C-3 26.8* 26.9* C-4 31.1** 31.1** C-5 31.0** 31.0**C-6 31.1** 31.1** C-7 26.9* 26.9* C-8 35.4 35.4 C-9 78.6 78.6 C-10 213.8213.8 C-11 38.4 38.4 C-12 123.0 123.0 C-13 133.5 133.4 C-14 27.5 27.5C-15 128.4 128.4 C-16 134.6 134.0 C-17 22.3 22.3 C-18 15.4 15.4 *, **exchangeable

Test Example A Evaluation of Plant Growth Promotion Effect of SpecificKetol Fatty Acid (I) (Growth Promotion Test)

1. Evaluation of Growth Promotion Effect on Morning Glory

Seeds of morning glory (variety: murasaki) (9 g) were subjected toconcentrated sulfuric acid treatment for 20 minutes, and then allowed tostand under water flow overnight. Subsequently, the seeds were placed onwet sea sand such that the hila of the seeds were directed upward for 24hours, to thereby produce roots. The seeds having roots were planted insea sand at a position 1.5-2.0 cm below the surface of the sand, andthen cultured under continuous light irradiation (for about five days).

The entire plant bodies of the morning glory having leaves producedthrough this culture were transferred into a culture solution [KNO₃,(250 mg), NH₄NO₃ (250 mg), KH2PO₄ (250 mg), MgSO₄.7H₂O (250 mg),MnSO₄.4H₂O (1 mg), Fe-citrate n-hydrate (6 mg), H₃BO₃ (2 mg), CuSO₄.5H₂O(0.1 mg), ZeSO₄.7H₂O (0.2 mg), Na₂MoO₄.2H₂O (0.2 mg), andCa(H₂PO₄)₂.2H₂O (250 mg) in distilled water (1,000 mL)].

Water or a 100 μM specific ketol fatty acid (I) aqueous solution wassprayed to the cultured morning glory, which was then placed in the darkovernight (for 14 hours). Thereafter, the morning glory was grown undercontinuous light irradiation at 25° C. for 16 days, and on the 16th daythe height of the plant was measured (N=8). The average of the heightsof the plants is shown in FIG. 1 [note: “I” shown in FIG. 1, refers to“specific ketol fatty acid (I)”; the same shall apply to thebelow-described Figs. ]. As is clear from FIG. 1, the plant of themorning glory becomes larger through application of specific ketol fattyacid (I).

2. Evaluation of Growth Promotion Effect on lettuce

One month after seeding of lettuce, spraying of a 50 μM specific ketolfatty acid (I) aqueous solution was carried out for five consecutivedays, and growth of the lettuce (plant width) was observed. The resultsare shown in FIG. 2. As shown in FIG. 2, specific ketol fatty acid (I)exerts the effect of promoting growth of the lettuce. The growthpromotion effect was maintained 48 days after the start of the test.

3. Evaluation of Growth Promotion Effect on Broad Bean

One month after seeding of broad bean, spraying of a 50 μM specificketol fatty acid (I) aqueous solution was carried out for fiveconsecutive days, and growth of the broad bean (plant width) wasobserved. The results are shown in FIG. 3.

As shown in FIG. 3, specific ketol fatty acid (I) exerts the effect ofpromoting growth of the broad bean. The growth promotion effect wasmaintained 48 days after the start of the test.

4. Evaluation of Growth Promotion Effect on Eustoma Russellianum

Three months after seeding of Eustoma russellianum, a 50 μM specificketol fatty acid (I) aqueous solution was sprayed to rosette leaves forfive consecutive days, and bolting was observed immediately after thespraying. Thereafter, growth of the plants of the Eustoma russellianumwas observed for 48 days. Although plant width did not increase as hadbeen expected, the plant height continued to increase 48 days afterobservation of growth. The results (plant height) are shown in FIG. 4.

5. Evaluation of Growth Promotion Effect on Cyclamen

Four months after seeding of cyclamen, spraying of a 50 μM specificketol fatty acid (I) aqueous solution was carried out for fiveconsecutive days. Thereafter, the plant width and the number of leaveswere observed for 48 days. The plant width and the number of leaves wereboth increased. The results are shown in FIG. 5.

6. Evaluation of Growth Promotion Effect on Digitalis

Two weeks after seeding of digitalis, spraying of an 80 μM specificketol fatty acid (I) aqueous solution was carried out for fiveconsecutive days. Furthermore, three months after the start of the test,spraying of an 80 μM specific ketol fatty acid (I) aqueous solution wascarried out for six weeks (once a week). Five-and-a-half months afterthe six-week spraying, the size of leaves and the plant height weremeasured. The results show that the size of leaves and the plant heightwere both increased (see FIG. 6).

7. Evaluation of Growth Promotion Effect on Chrysanthemum

Two weeks after seeding of chrysanthemum, spraying of an 80 μM specificketol fatty acid (I) aqueous solution was carried out for fiveconsecutive days. Furthermore, three months after the start of the test,spraying of an 80 μM specific ketol fatty acid (I) aqueous solution wascarried out for six weeks (once a week). Bolting was not observed at anutrition growth stage of the chrysanthemum. Four months after the lastspraying, the plant width was measured. The results show that the plantwidth of the chrysanthemum is increased significantly (see FIG. 7).

8. Evaluation Growth Promotion Effect on Geranium

Two weeks after seeding of geranium, spraying of an 80 μM specific ketolfatty acid (I) aqueous solution was carried out for five consecutivedays. Furthermore, three months after the start of the test, spraying ofan 80 μM specific ketol fatty acid (I) aqueous solution was carried outfor six weeks (once a week). Two types of geranium; i.e., geraniumhaving mottled leaves and geranium having leaves of no mottles, weresubjected to the test. Five-and-a-half months after the last spraying,the size of the leaves was measured. The results show that growth of theleaves of these two types is promoted (see FIG. 8).

9. Evaluation of Growth Promotion Effect on Primula Melacoides

One-and-a-half months after seeding of Primula melacoides, spraying ofan 80 μM specific ketol fatty acid (I) aqueous solution was carried outfor five consecutive days. Furthermore, four months after the start ofthe test, spraying of an 80 μM specific ketol fatty acid (I) aqueoussolution was carried out for six weeks (once a week). Bolting was notobserved at a nutrition growth stage of the Primula melacoides.Six-and-a-half months after the last spraying, the plant width and theleave size were measured. The results show that the plant width andleave size of the chrysanthemum are increased (see FIG. 9).

10. Evaluation of Growth Promotion Effect on Begonia Sempaflorens

Two weeks after seeding of Begonia sempaflorens, spraying of an 80 μMspecific ketol fatty acid (I) aqueous solution was carried out for fiveconsecutive days. Furthermore, three months after the start of the test,spraying of an 80 μM specific ketol fatty acid (I) aqueous solution wascarried out for six weeks (once a week). Four months after the lastspraying, the leave size was measured. The results show that growth ofthe leaves is promoted (see FIG. 10).

11. Evaluating of Growth Promotion Effect on Dianthus Caryophyllus

Seedlings of Dianthus caryophyllus (feeling scarlet) were planted inearly October, and then grown by means of a customary method. Inmid-April of the next year, spraying of a 100 μM specific ketol fattyacid (I) aqueous solution was carried out (5 mL per plant), and then theheight of the plants was measured. The results show that growth of theDianthus caryophyllus plant was promoted in the group to which thespecific ketol fatty acid (I) had been applied, although spraying of thespecific ketol fatty acid (I) had been carried out only once (see FIG.11).

12. Evaluation of Growth Promotion Controlling Effect on Oryza Sativa L.

(1) Good-quality seeds of Oryza sativa L. (variety: koshihikari) (200 g)were immersed in water (800 mL) at 10° C. for 13 days. Thereafter, theseeds were equally divided into four groups, and the respective seedgroups were immersed in specific ketol fatty acid (I) aqueous solutions(concentration: 0 μM, 1 μM, 10 μM, and 100 μM) (200 mL) at 30° C. for1.5 days. The immersed seeds were planted in a four-divided seedbedtray, and grown under no light irradiation at 2720 C. for three days.Subsequently, the grown seedlings were exposed to the typical outsideenvironment.

Six days after the exposure, 18 seedlings were randomly selected fromeach group, and the heights of the seedlings were measured, and thenaveraged. The results are shown in FIG. 12. As shown in FIG. 12, thedegree of growth promotion of the seedlings regarding the height iscommensurate with the application amount of the specific ketol fattyacid (I).

Thus, the plant growth promotion effect of the present plant activator,which is confirmed in the aforementioned tests employing various plants,is also observed in Oryza sativa L.

Subsequently, in consideration of:practical handling of seedlings ofOryza sativa L., the effect of application of specific ketol fatty acid(I) was evaluated. Specifically, whether or not specific ketol fattyacid (I) exerts the effect of controlling growth of the third leaves ofseedlings of Oryza sativa L. was evaluated, since the time when thethird leaves are grown in seedlings of Oryza sativa L. is considered tobe a suitable time for transferring the seedlings from a seedbed to apaddy field. In order to perform this evaluation, three weeks after theaforementioned light irradiation treatment, seedlings of each group wererandomly selected, and the average of the proportions of the secondleaves and third leaves was obtained. The results are shown in FIG. 13.As shown in FIG. 13, specific ketol fatty acid (I) exerts the effect ofcontrolling growth of the third leaves. However, unlike the case ofpromotion of growth of the seedlings, the optimum concentration of thespecific ketol fatty acid (I) aqueous solution is 1 μM.

The results show that, when specific ketol fatty acid (I) is used as anactive ingredient of the present plant activator in order to shorten thegrowth period of seedlings of Oryza sativa L. in a seedbed, theapplication amount of the specific ketol fatty acid (I) must bedetermined appropriately.

(2) Seedlings of Oryza sativa L. were grown in a seedbed in a mannersimilar to that described in (1), except that the seedlings wereimmersed in ion-exchange water at 10° C. for 15 days, withoutapplication of specific ketol fatty acid (I) described above in (1).Subsequently, the resultant seedlings which had been divided into fivegroups, each group containing three subgroups (only control groupcontaining four subgroups) and each subgroup containing 16 seedlings,were exposed to the outside environment. Immediately after thisexposure, spraying of specific ketol fatty acid (I) was carried out (0ppm for a first group (control group), 25 ppm for second and thirdgroups, 50 ppm for fourth and fifth groups). Thirty days after thespraying, the seedlings were planted in a paddy field, and additionalspraying of specific ketol fatty acid (I) (25 ppm) was carried out fortwo groups; i.e., the third group (total amount of the acid (I): 25+25ppm) and the fifth group (total amount of the acid (I): 50+25ppm).

Subsequently, the seedlings of the Oryza sativa L. were grown in thepaddy field by means of a customary method. Forty-one days after theabove planting, in each group, the plant height and the number of stemsper plant (at a section at which four seedlings were planted) weremeasured, and then averaged.

The plant height was 56 cm in the control group (first group), 57 cm inthe second group (amount of the specific ketol fatty acid (I): 25 ppm),58 cm in the fourth group (amount of the specific ketol fatty acid (I):50 ppm), 57 cm in the third group (amount of the specific ketol fattyacid (I): 25+25 ppm), and 58 cm in the fifth group (amount of thespecific ketol fatty acid (I): 50+25 ppm) Therefore, significantdifference was not observed between these groups.

The number of stems per plant was 34 in the control group (first group),38 in the second group (amount of the specific ketol fatty acid (I): 25ppm), 38 in the fourth group (amount of the specific ketol fatty acid(I). 50 ppm). 39 in the third group (amount of the specific ketol fattyacid (I): 25+25 ppm), and 37 in the fifth group (amount of the specificketol fatty acid (I): 50+25 ppm) Briefly, the number of stems per plantin each of the second through fifth groups was about 10% greater thanthat of stems per plant in the control group. However, differenceattributed to the application manner of the specific ketol fatty acid(I) was not observed.

The results show that specific ketol fatty acid (I) serving as an activeingredient of the present plant activator exerts the effect ofcontrolling growth of Oryza sativa L.; i.e., the effect of increasingthe number of stems. Therefore, the specific ketol fatty acid (I) exertsthe effect of increasing the yield of rice on the basis of unit area ofthe paddy field in which the seedlings are planted; i.e., anconsiderably important effect in production of rice.

The results of the aforementioned growth promotion tests show thatspecific ketol fatty acid (I) exerts excellent effect of promotinggrowth of various forms of many plants. Particularly, the specific ketolfatty acid (I) exerts the effect of promoting growth of a plant in anearly stage of its growth, and the growth promotion effect iscontinuous.

Thus, it is apparent that specific ketol fatty acid (I) serving as anactive ingredient of the present plant activator exerts the effect ofpromoting growth of various plants, and the present plant activator isuseful.

As described above, it is clear that the present plant activator can beused as a plant growth promoting agent or a plant growth controllingagent.

Test Example B Evaluation of Plant Dormancy Preventive Effect ofSpecific Ketol Fatty Acid (I) (Plant Dormancy Preventive Test)

When strawberry seedlings are exposed directly to low temperatureconditions in winter, the seedlings enter dormancy, and growth of theseedlings is stopped. Whether or not the present plant activator exertsthe effect of preventing dormancy was evaluated.

Specific ketol fatty acid (I) aqueous solutions [concentration; 10 μM,100 μM, and 0 μM (control)] were applied through spraying to strawberryseedlings on August 27 (at day 0), September 3, and September 8.Thereafter, the seedlings were cultured outdoors without artificialtreatment such as low temperature treatment, and formation of flowerbuds was observed with passage of time. In the control group, no flowerbud formation was observed. In contrast, in the groups in which thespecific ketol fatty acid (I) was applied through spraying, flower budformation proceeded, and the number of flowers increased (this flowerbud formation promotion effect agrees with the description of JapanesePatent Application Laid-Open (kokai) No. 11-29410).

At the one hundred and eighth day, percent dormancy (percentage ofdormant plants—which are plants in which growth of small leaf buds withmarkings is not observed 15 days after a marking was applied to the leafbuds—with respect to the entirety of the test plants) was measured. As aresult, in the control group, dormancy of the entire plants wasobserved. In contrast, in the groups in which the specific ketol fattyacid (I) aqueous solution was applied through spraying, dormancy ofstrawberry was prevented. The results show that the dormancy preventiveeffect is more significant in the group in which the specific ketolfatty acid (I) of low concentration (10 μM) was applied than in thegroup in which the fatty acid (I) of high concentration (100 μM) wasapplied (see FIG. 14).

The results show that when the present plant activator of lowconcentration is applied to a plant, the activator exerts the effect ofpreventing dormancy of the plant; the activator can be used as a plantdormancy preventive agent or a plant growth controlling agent; and theactivator is useful.

Test Example C Evaluation of Plant Stress (Dry Stress) SuppressiveEffect of Specific Ketol Fatty Acid (I)

Seeds of lettuce (50 seeds per test group) were immersed in specificketol fatty acid (I) aqueous solutions [concentration: 2 μM, 10 μM, 20μM, and 0 μM (control)] for 72 hours, and dried in air for 48 hours. Theresultant seeds were disposed on water-containing filter paper, andallowed to germinate. In each test group, germination rate—percentage(%) of germinated seeds with respect to the entirety of seeds—wasobtained.

The results are shown in Table 2.

TABLE 2 Specific ketol Germination fatty acid (I) rate Number ofgerminated concentration (μM) (%: n = 50) seeds 0 10 5 2 86 43 10 98 4920 90 45

As is apparent from the results, in the control group, most seeds failedto endure the dry stress in the drying step, resulting in failure ofgermination. In contrast, most of the seeds which had been immersed inthe specific ketol fatty acid (I) aqueous solution successfullygerminated.

From the above results, it is clear that the present plant activatorexerts the effect of enhancing tolerance of plants against dry stress;the activator can be used as a plant stress suppressing agent or a plantgrowth controlling agent; and the activator is useful.

Test Example D Growth Controlling Effect of Specific Ketol Fatty Acid(I) on Fungi

(1) Evaluation of Effect of Proliferating Hyphae of P. citrinopileatusSing. (Edible Mushroom) Belonging to Pleurotus of Basidiomycota

A potato-dextrose-agar culture medium was sterilized by use of anautoclave. After the medium was cooled to a temperature at which theagar was not solidified, a 1 mM specific ketol fatty acid (I) aqueoussolution which had been sterilized by use of a membrane filter was addedto the medium, to thereby prepare four culture media; i.e., a culturemedium containing 0 μM of the fatty acid (I), a culture mediumcontaining 10 μM of the fatty acid (I), a culture medium containing 30μM of the fatty acid (I), and a culture medium containing 100 μM of thefatty acid (I). Each of the culture media was solidified in a 10-cmplate, and then hyphae of P. citrinopileatus (one platinum loop) wasinoculated on the medium. Subsequently, the hyphae were cultured at 37°C., and proliferation of the hyphae was observed (10 plates for eachgroup). Proliferation of the hyphae was evaluated on the basis of theaverage of the diameters of the proliferated hyphae on the plate. Theresults are shown in FIG. 15. As is clear from FIG. 15, the degree ofproliferation of the hyphae of P. citrinopileatus is dependent on theconcentration of the specific ketol fatty acid (I).

(2) Evaluation of Growth Promotion Effect on Carpophore of LentinusEdodes (Berk.) Singer

Wood (Quercus serrata) containing hyphae of Lentinus edodes was cut intopieces having a length of about 15 cm, and the pieces were immersed in10° C. water for 24 hours, after which the pieces were allowed to standin a container of high humidity. Subsequently, specific ketol fatty acid(I) aqueous solutions (concentration: 0 μM, 3 μM, 30 μM, and 100 μM)were applied through spraying to the resultant pieces (six pieces foreach group). Each solution was applied to each group (5 mL for eachpiece). Subsequently, the carpophores of Lentinus edodes were culturedin the same container at 18° C. under weak light irradiation. Thisculture was continued for five days, and then growth of the carpophoresof Lentinus edodes was observed. FIG. 16 is a photograph showing thecarpophores of Lentinus edodes cultured in the groups (note: {circlearound (1)} application of the specific ketol fatty acid (I) (0 μm),{circle around (2)} (3 μM), {circle around (3)} (30 μM), and {circlearound (4)} (100 μM)). The average of the carpophores per piece was 0 inthe 0 μM application group, 0.17 in the 3 μM application group, 1.0 inthe 30 μM application group, and 1.0 in the 100 μM application group.

The results show that specific ketol fatty acid (I) exerts the effect ofpromoting growth of carpophores of Lentinus edodes during culture.

The results of the aforementioned tests (1) and (2) show that whenspecific ketol fatty acid (I) is applied to ascomycete or basidiomycete,proliferation of hyphae thereof can be promoted, and harvest efficiencyof carpophores can be enhanced. Furthermore, the present plant activatormay contribute to establishment of an artificial culture method ofmushrooms which at present are difficult to culture artificially (e.g.,Tricholoma matsutake).

INDUSTRIAL APPLICABILITY

As described above, the present invention provides a plant activatorexerting excellent effect of controlling growth of various plants.

1. A method for promoting growth of a fungus comprising applying to thefungus a C₄-C₂₄ ketol fatty acid.
 2. The method of claim 1, whereinhyphae of the fungus are proliferated.
 3. The method according to claim1, wherein the C₄-C₂₄ ketol fatty acid contains a carbon atomconstituting a carbonyl group and a carbon atom connected to a hydroxylgroup, one of the above carbon atoms being located at the α or γposition with respect to the other carbon atom.
 4. The method of claim1, wherein the C₄-C₂₄ ketol fatty acid contains one to six carbon-carbondouble bonds, such that the number of the double bonds does not exceedthe number of carbon-carbon bonds in the ketol fatty acid.
 5. The methodof claim 1, wherein the ketol fatty acid contains 18 carbon atoms, andtwo carbon-carbon double bonds.
 6. The method of claim 1, wherein theC₄-C₂₄ ketol fatty acid is 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoicacid.